Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod- borne infectious agent in the United States, and contributes to a significant amount of morbidity in persistently infected patients. B. burgdorferi is effective at colonizing both mammalian and arthropod hosts and, as such, must modulate gene expression quickly to adapt to these different environments. Although we know some of the molecular signals that alter gene expression in B. burgdorferi, we still understand little regarding how potential virulence determinants are regulated in this pathogen. In the past funding period, we have characterized a regulatory protein, designated BosR, which is involved in regulating the oxidative stress response in B. burgdorferi. Recently, we found that two genes linked to bosR, bb0646 and bb0648, share a transcript and thus comprise an operon. These genes encode for an exported lipase (bb0646) and a serine/threonine kinase (bb0648), respectively, which we suggest are involved in the oxidative stress response in B. burgdorferi. The central hypothesis is that BosR, and the linked genes bb0646 and bb0648, coordinate an important adaptive response that senses the redox status of the cell. To address this hypothesis, we propose the following Specific Aims: (1) Characterize the bosR operon in infectious B. burgdorferi. The working hypothesis is that bosR and its flanking genes, bb0646 and bb0648, respond appropriately to the redox status of the cell to combat toxic oxidizing compounds generated during the arthropod blood meal or the mammalian innate immune response. We have not yet been able to evaluate the role of BosR in infectious isolates, presumably since bosR regulates essential genes. Here we will use a recently developed tightly regulated inducible system to generate a conditional mutant in bosR in infectious B. burgdorferi; (2) Assess the infectivity deficit in conditional mutants and knockouts in BosR-regulated genes. The working hypothesis is that genes regulated by BosR are required for physiologically important processes related to oxidative stress and infectivity; (3) Determine the mechanism of BosR-mediated regulation. The working hypothesis is that BosR alters its regulatory activity via oxidation and metal binding, which changes its avidity for target sequences; and (4) Decipher the role of BB0646 and BB0648 in B. burgdorferi pathogenesis. Our working hypothesis is that both of these gene products are involved in host adaptation by modifying polyunsaturated lipid substrates and coordinating a global response to oxidative stress, respectively. The information from these studies will provide insight into how B. burgdorferi adapts to the redox status of the host via BosR, BB0646, and BB0648, and will help to determine how the ensuing response relates to the disease potential of this important pathogen. PUBLIC HEALTH RELEVANCE: Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod-borne infectious agent in the United States, and thus is an important Public Health issue. The studies described herein are designed to address how B. burgdorferi is able to adapt to both ticks and mammals, in the context of host mediated oxidation defenses, and how this adaptive response affects the ability of this bacterium to persist within the environment and cause disease.